JP2012509750A - Functional titanium implants and similar renewable materials - Google Patents

Abstract

A surface treated to increase protein absorption, promote osteoblast migration, promote osteoblast adhesion, promote osteoblast diffusion, promote osteoblast proliferation, and promote osteoblast differentiation. Implant.
A method for imparting functionality to an implant, wherein the surface of the implant is treated to carry a positive charge. The implant has improved tissue-implant integration and / or bone-implant integration.
[Selection] Figure 1

Description

This application claims priority based on US patent application Ser. No. 61 / 117,831 filed Nov. 25, 2008, the entire contents of which are incorporated by reference.
The present invention relates to a medical implant used for biomedical.

  Femoral neck fractures due to osteoporosis and degenerative changes in the knee and hip joints are common problems. In the United States, more than 500,000 hip and knee joint reconstruction operations are performed annually, and a titanium implant as an anchor is an important therapeutic means for this operation. Fractures in these areas are essentially unacceptable to fix bones (eg, fixation support), and implants are usually permanent and active due to daily activities such as gravity and walking immediately after surgery. And / or exposed to periodic impacts. Problems with the results of such operations include alarming disability, long-term assistance, mortality, corrective surgery that occurs at a relatively high rate of 5-50%, and a practical quality of life decline. Is mentioned.

  As another determinant, the placement of implants used for these purposes inhibits the ability of bone to regenerate and causes metabolic activities such as osteoporosis and aging that delays bone repair around the implant. Thus, to minimize morbidity and maximize functional recovery and long-term prognosis, we have never been more committed to establishing a quick and firm bone and joint anchor using intraosseous implants. Yes.

  On the other hand, the use of dental titanium implants as a regenerative operation for missing teeth is widely recognized. However, various risks exist for dental implant treatment, including the quality and size of the patient's teeth, overall condition, and age. Furthermore, it takes a long time (4-6 months) for the titanium implant to be integrated with the bone and to withstand the occlusal load, thus substantially limiting these beneficial treatments. Implants with improved osteogenesis (osteoconductivity) capabilities are very beneficial to patients and dentists.

  In addition to bone, tissue regeneration therapies other than bone, joint and tooth reconstruction treatments face many problems. For example, currently practiced treatments for bones after injury and degenerative changes require the use of biomolecules such as growth factors for tissue regeneration. However, the effects of biomolecules and the amount of bone that can be regenerated are still limited. The side effects due to biomolecules and the cost required for treatment are also significant.

  Implants with improved bioactivity when supplied with a carrier biomaterial have the potential to be used to promote biological reactions required for tissue regeneration.

  Described herein is a medical implant having a metal surface, wherein the metal surface comprises a positively charged metal oxide. The metal may be titanium, gold, platinum, tantalum, niobium, nickel, iron, chromium, cobalt, zirconium, aluminum, and palladium. In one embodiment, the implant may have a metallic or non-metallic carrier material.

In one embodiment, the medical implant has a titanium surface. Titanium surface has a TiO _2. In one embodiment, the titanium surface is substantially free of hydrocarbons.

  The implant surface can attract proteins and / or cells at an accelerated rate. The protein may use bovine serum albumin, fraction V, and bovine plasma fibronectin. The cells may be human mesenchymal stem cells and osteoblasts. Proteins or cells may be directly attached to the treated implant surface, for example, without using a divalent cation for crosslinking.

  The implant surface can cause or enhance tissue-implant integration and / or bone-implant integration. The implant surface can be the following or a combination of: increased protein absorption, promoted osteoblast migration, promoted osteoblast adhesion, promoted osteoblast proliferation, promoted osteoblast proliferation, and osteoblast Promotion of cell differentiation.

  Here, a method for imparting functionality to a medical implant is described, the method comprising: (1) providing a metal implant surface; and (2) treating the implant surface to a positively charged surface. Or enhancing the positive charge on the surface. In some embodiments, the method is positively charged in a physiological environment. The physiological environment may have a pH of about 7. In some embodiments, the method can charge the surface positively even if the pH is less than 7 or greater than 7.

  In one embodiment, the treated surface can adsorb proteins and / or cells at an improved rate compared to an untreated surface.

  In one embodiment, the implant has a titanium surface. In one embodiment, the titanium surface has titanium dioxide.

  In one embodiment, the implant surface is treated by irradiating with ultraviolet light (UV radiation). UV radiation can be irradiated using a UV lamp. The UV radiation may have a wavelength of about 10 nm to 400 nm. In some embodiments, the UV radiation may have a wavelength of about 170 nm to about 270 nm or about 340 nm to 380 nm. In some embodiments, the surface is treated by irradiating a combination of UV light having a wavelength of about 170 nm to about 270 nm and UV light of about 340 nm to about 380 nm.

The intensity of UV radiation may be in a wide range. For example, the UV ray intensity may be in the range of 0.001 mW / cm ^{2 to} 100 mW / cm ² . In some embodiments, the UV radiation may be about 0.1 mW / cm ² intensity, or about ² mW / cm ² . The treatment with UV rays may be within 48 hours, for example, 30 seconds, 1 minute, 5 minutes, 15 minutes, 30 minutes, 1 hour, 5 hours, 10 hours, 24 hours, 36 hours, and 48 hours. is there.

  In one embodiment, the method further comprises processing the implant surface prior to applying the treatment to the implant surface. The implant surface may be subjected to physical processing or chemical processing. The physical processing may be machining or sand blasting. The chemical processing may be etching with acid or alkali. The acid may be sulfuric acid. The processing surface may be positively charged. UV radiation treatment helps to impart a positive charge to the processed surface.

  In some embodiments, the treated surface has a metal oxide cation. The metal oxide cation may be a titanium oxide cation.

In some embodiments, the treated implant surface can adsorb bovine serum albumin, fraction V, bovine plasma fibronectin, and the like. In one embodiment, the treated implant surface can attract human mesenchymal stem cells and osteoblasts. The protein or cell may adhere directly to the treated implant surface without using, for example, a divalent cation for crosslinking. In one embodiment, the treated titanium surface does not have a divalent cation such as Ca ²⁺ , Mg ²⁺ , Zn ²⁺ .

  The treated implant surface can improve tissue-implant integration and / or bone-implant integration at the implant site. The treated implant surface has a better bone forming ability than the untreated implant surface. The implant surface can be the following or a combination of: increased protein absorption, promoted osteoblast migration, promoted osteoblast adhesion, promoted osteoblast spread, promoted osteoblast proliferation, and osteoblasts Induction of differentiation.

  The methods described above can be used to increase the osteogenic activity of the implant, increase the osteoconductive ability of the implant, and improve tissue-implant and / or bone-implant integration.

  Described herein is a medical implant having functionality on its surface by the method described above.

FIG. 1 shows the initial bioactivity of the acid etched titanium surface at different times and with and without UV irradiation. FIG. 1A shows 2 hours and 24 hours when the titanium disk is used immediately after being newly processed, after 4 weeks (stored in a dark room at room temperature), and when treated with UV after 4 weeks. The mean value of bovine serum albumin absorption rate ± SD (standard deviation) after 72 hours of culture is shown. FIG. 1B shows the amount of human mesenchymal stem cells (MSCs) that migrated through 8 μm pores during 3 hours of culture on acid-etched titanium discs under different conditions. FIG. 1C shows human MSCs adhered to titanium discs evaluated by WST-1 detection at 3 and 24 hours after inoculation. In all panels, data are shown as mean ± SD (n = 3). Figure 2 shows the initial dispersal and cytoskeletal changes of human mesenchymal stem cells 3 hours after inoculation on acid-etched titanium surfaces under different conditions. Different conditions: freshly processed surface, surface after 4 weeks, surface after 4 weeks treated with UV light. FIG. 2A shows a confocal microscope image of cells stained with actin filaments (red), anti-paxillin (green), or combinations thereof with rhodamine phalloidin. FIG. 2B is an evaluation of cell morphometry performed using these images. Data are shown as standard values ± SD (n = 10). Fig. 3 shows that the biomechanical push-in test was evaluated, and the bone-titanium integration of the newly processed titanium surface and the titanium surface etched with UV-treated acid is compared to the surface after 4 weeks. Indicates that it is being promoted. The push-in values for machined and acid etched implants are described with and without UV radiation treatment. Data are shown as mean ± SD (n = 5). FIG. 4 shows the improvement in albumin absorption A and cell adhesion B on the positively charged titanium surface. FIG. 4A shows various titanium surfaces (freshly processed titanium surface, 4 weeks later titanium surface, 4 weeks UV treated titanium surface) with and without ion treatment for 24 hours prior to albumin culture. Absorption of albumin during 3 hours of culture is shown. The medium for albumin culture was adjusted to pH 7 or 3. FIG. 4B shows the amount of human MSCs adhered to various titanium surfaces in a 24 hour culture. The titanium surface was prepared in the same manner as in FIG. 4A. The medium was adjusted to pH7. Data are shown as mean ± SD (n = 3). FIG. 5 shows a newly elucidated state in which proteins and cells are electrostatically adhered to the titanium surface. The left side (old titanium) and the right side (new titanium or UV-treated titanium) each do not attract cells and the other attracts cells. FIG. 6 is a generalization of the enhanced bioactivity of a newly processed titanium surface and a UV-treated titanium surface. FIG. 6A is a 6 hour albumin-absorbing culture on freshly processed, 4 week, 4 week later machined and sandblasted titanium discs that had been UV-irradiated. Data are shown as mean ± SD (n = 3). FIG. 6B shows a 6 hour fibronectin absorption culture on freshly processed, 4 weeks, 4 weeks later machined and sandblasted titanium discs. Data are shown as mean ± SD (n = 3). FIG. 6C shows the bone-titanium integration measured by a push-in test of the machined implant with and without UV treatment. Data are shown as mean ± SD (n = 5). Figures 7A-G show osteoblast affinity for titanium surfaces induced by ultraviolet light (UV radiation). Two different forms of surface were provided, a machined surface and an acid etched surface. FIG. 7A shows the superhydrophilic titanium surface obtained after 48 hours of UV radiation treatment (left figure). The change in hydrophilicity was evaluated by the contact angle of H ₂ O after UV treatment for various times (line graph). FIG. 7B shows the decrease in hydrophilicity of the titanium surface in the dark after the 48 hour UV radiation treatment has stopped. FIGS. 7C and D show the rate of protein absorption on the titanium surface with and without pretreatment with UV radiation. Albumin (C) and fibronectin (D) were cultured on the titanium surface for 2, 6, and 24 hours. FIGS. 7C and D show the rate of protein absorption on the titanium surface with and without pretreatment with UV radiation. Albumin (C) and fibronectin (D) were cultured on the titanium surface for 2, 6, and 24 hours. FIG. 7E shows an evaluation by WST-1 color measurement of the relative number of osteoblasts attached to the titanium surface with and without UV treatment after 3 and 24 hours of culture. FIGS. 7F and G show the time-dependent rate of change of the titanium affinity UV treatment for proteins and osteoblasts. The albumin absorption rate (F) and osteoblast adhesion rate (G) on the titanium surface versus time of pretreatment with UV rays are plotted. The data from FIGS. 7C to 7G are shown as mean values ± SD (n = 3), and ^** p value <. 01 and ^* p value <. There was a significant difference at 05. FIGS. 7F and G show the time-dependent rate of change of the titanium affinity UV treatment for proteins and osteoblasts. The albumin absorption rate (F) and osteoblast adhesion rate (G) on the titanium surface versus time of pretreatment with UV rays are plotted. The data from FIGS. 7C to 7G are shown as mean values ± SD (n = 3), and ^** p value <. 01 and ^* p value <. There was a significant difference at 05. FIGS. 8A-C show an early aspect of osteoblasts on UV-treated titanium. FIG. 8A shows the initial osteoblast distribution and cytoskeletal organization on the titanium surface with and without pretreatment with UV radiation. It is a confocal microscope image of double-stained osteoblast nuclei (blue) with DAPI and actin filaments (red) with rhodamine phalloidin 3 hours after inoculation. The bar is 10 μm. The images were used to evaluate cell morphometry (histograms are shown below). Data are shown as mean ± SD (n = 6), ^* p value <0.05, and there was a significant difference between the UV irradiated and untreated control surfaces. FIG. 8B shows the density of osteoblasts on the 2nd and 5th day of culturing on the titanium surface with and without UV treatment (histogram below). The fluorescence image of the cells obtained on the second day is shown at the top so that the cell density can be confirmed. FIG. 8C evaluated the osteoblast proliferation activity on the titanium substrate by incorporating BrdU into each cell of the second day culture. The data in FIGS. 8B and C are shown as mean values ± SD (n = 3), and ^** p value < ^{.P on} the UV-treated surface and untreated control surface, respectively. 01 and ^* p value <. There was a significant difference at 05. Figures 9A-D show enhanced osteoblast phenotype and enhanced differentiation on UV-treated titanium surfaces. FIG. 9A shows the UV-stimulated activity of alkaline phosphotase (ALP), a marker for early stages of osteoblasts. In the upper figure, ALP staining of osteoblasts cultured for about 10 days on a titanium substrate is shown. The ALP positive area is shown as a percentage in the culture area (lower left histogram). Also shown is colorimetric quantification of ALP activity normalized for each cell (lower right histogram). FIG. 9B shows the mineralization ability (late stage marker) of osteoblasts. The upper figure is a von Kossa staining of osteoblasts after 14 days of culture. The von Kossa positive area in the culture area is shown as a percentage (lower left histogram). Also shown is the total amount of calcium deposited by color measurement (lower right histogram). FIGS. 9C and D show the expression of bone-related genes in osteoblasts cultured on machined titanium surfaces (C) and acid-etched titanium surfaces (D). Osteoblasts were cultured on UV-treated titanium and non-UV-treated titanium, and gene expression was semi-quantified and evaluated by reverse transcription polymerase chain reaction (RT-PCR). The image of electrophoresis is shown in the upper figure. The quantitative level of gene expression relative to the expression level of GAPDH mRNA is shown below. C: Untreated control. UV: UV irradiation treatment. The data from FIGS. 9A to 9D are shown as mean values ± SD (n = 3), and the ^** p value <. 01 and ^* p value <. There was a significant difference at 05. FIGS. 9C and D show the expression of bone-related genes in osteoblasts cultured on machined titanium surfaces (C) and acid-etched titanium surfaces (D). Osteoblasts were cultured on UV-treated titanium and non-UV-treated titanium, and gene expression was semi-quantified and evaluated by reverse transcription polymerase chain reaction (RT-PCR). The image of electrophoresis is shown in the upper figure. The quantitative level of gene expression relative to the expression level of GAPDH mRNA is shown below. C: Untreated control. UV: UV irradiation treatment. The data from FIGS. 9A to 9D are shown as mean values ± SD (n = 3), and the ^** p value <. 01 and ^* p value <. There was a significant difference at 05. FIG. 10 shows bone-titanium integration improved by UV radiation treatment evaluated by biomechanical push-in test. The push-in values for machined and acid etched implants are described with and without UV treatment. Data are shown as mean ± SD (n = 5). On the surface treated with UV radiation and the untreated control surface, ^** p value <. 01 and ^* p value <. There was a significant difference at 05. FIG. 11 shows bone formation around the implant periphery promoted by UV radiation. 11A-D are original magnifications of 40 times, FIGS. 11E-H are 200 times, FIGS. 11I-L are 400 times, and cell images of titanium implants etched with Goldner trichrome-stained acid. Show. In addition, the UV-treated implant at 2 weeks undergoes intense bone formation that prevents soft tissue from forming between the bone and the implant (arrow in FIG. 11F), and this is because bone is directly deposited on the implant surface. (Fig. 11J arrow). In contrast, the bone around the untreated control is fragmented (FIG. 11E) and contains soft tissue moving between the bone and the implant surface, which is a direct bone-implant surface contact. (Arrow in FIG. 11I). Such morphological differences at the implant and bone interface are also apparent at 4 weeks (FIGS. 11G and H). Extensive bone dispersion along the implant surface without being inhibited by soft tissue has been observed around UV-treated implants (FIGS. 11H and L), while much of the bone around untreated implants is Separated from the implant surface by soft tissue (arrows in FIGS. 11G and 11K). Shows average cell morphometry of bone-implant contact (FIG. 11M), bone mass in proximal region (FIG. 11N), bone mass in distal region (FIG. 11O) and soft tissue inhibition (FIG. 11P). . On the surface treated with UV radiation and the untreated control surface, ^** p value <. 01 and ^* p value <. There was a significant difference at 05. 40X image of bone formation at the second week (W2) of the periphery of the implant treated with UV radiation. 40X image of 4 week (W4) bone formation on an untreated control implant surface. Image at 40x magnification of 4 weeks (W4) bone formation around the UV-treated implant periphery. Image at 200x magnification of bone formation at the periphery of the untreated control. Image at 200x magnification of the 2 weeks of bone formation around the periphery of the UV treated implant. Image at 200 magnification at 4 weeks of bone formation around the periphery of an untreated control implant. Image at 200 magnification of 4 weeks of bone formation at the periphery of the implant treated with UV radiation. Image at 400X of soft tissue inhibition at the periphery of an untreated control implant. Image at 400x magnification of direct bone deposition of UV treated implant. 400X image of untreated control implant bone separated from the implant surface by soft tissue. 400X image of extensive bone dispersion of UV treated implant. Average cell morphometry of bone-implant contact. Bone mass in the proximal region. Bone mass in the distal region. Inhibition by soft tissue. Figures 12A-M show changes in the properties of titanium surfaces with biological effects induced by UV radiation. FIG. 12A shows an X-ray diffraction (XRD) spectrum of a machined titanium surface and an acid etched titanium surface, a TiO ₂ pure rutile structure produced by heating at 923 K and 673 K, and rutile The mixed structure of anatase is shown. FIG. 12B shows the light absorption spectrum of a machined titanium surface and an acid etched titanium surface. FIG. 12C shows X-ray photoelectron spectroscopy (XPS) of a machined titanium surface and an acid etched titanium surface. FIG. 12D is an enlarged view of the XPSTi2p peak of FIG. 12C. FIGS. 12E-12G show changes in the XPS state of Ti2p (FIG. 12E), Ols (FIG. 12F), and Cls (FIG. 12G) on titanium surfaces etched with acid after irradiation with UV radiation for various times. FIGS. 12E-12G show changes in the XPS state of Ti2p (FIG. 12E), Ols (FIG. 12F), and Cls (FIG. 12G) on titanium surfaces etched with acid after irradiation with UV radiation for various times. FIGS. 12E-12G show changes in the XPS state of Ti2p (FIG. 12E), Ols (FIG. 12F), and Cls (FIG. 12G) on titanium surfaces etched with acid after irradiation with UV radiation for various times. FIG. 12H shows the change in the percentage of atoms on the titanium surface etched by acid treated with UV radiation for different times in percentage. FIG. 12I is a plot of albumin absorption after 3 hours of incubation versus acid-etched titanium surface carbon atoms, indicating a significant inverse correlation. FIG. 12J is a plot of osteoblast adhesion after 3 hours of incubation versus acid-etched titanium surface carbon atoms, indicating a significant exponential relationship. FIGS. 12K and 12L are plots of albumin resorption rate (FIG. 12K) and osteoblast adhesion rate (FIG. 12L) against the contact angle of H ₂ O on an acid etched titanium surface. It shows that there is no particular relevance. FIGS. 12K and 12L are plots of albumin resorption rate (FIG. 12K) and osteoblast adhesion rate (FIG. 12L) against the contact angle of H ₂ O on an acid etched titanium surface. It shows that there is no particular relevance. FIG. 12M shows a schematic representation of imparting TiO ₂ photofunctionality of biocompatible TiO ₂ by light formation that accelerates and improves protein absorption and osteoblast adhesion and dispersion. FIG. 13 shows the number of cells attached to the titanium surface variously treated with UV radiation.

Described herein a medical implant having a metal surface, a medical implant having a metal surface, the metal surface has a metal oxide positively charged. The metal may be titanium, gold, platinum, tantalum, niobium, nickel, iron, chromium, cobalt, zirconium, aluminum, and palladium. In some embodiments, the metal surface has a metal oxide cation.

Titanium surface Since the titanium surface is considered to be negatively charged, cations react with the titanium surface, such as Ca ²⁺ . On the other hand, many proteins and biological cells are negatively charged under physiological conditions and can repel titanium surfaces.

  Titanium implants are used as reconstruction anchors in orthopedic and dental illnesses and problems. The success of implant anchoring involves the scale of bone deposition directly on the titanium surface without being inhibited by soft / connective tissue. This is called “bone-implant integration” or “osseointegration”. Current orthopedic and dental titanium implants have been developed based on this concept and are called “Osseointegrated implants”. However, the total implant area covered by bone remains at 45 ± 16% or 50-75%, well below the ideal 100%. Many implants fail due to incomplete placement or early or late destructive changes at the bone-implant interface. The reason why bone tissue does not form all around the implant is unknown.

Titanium oxide (TiO ₂ ) ultraviolet (UV radiation) induced superhydrophilicity was discovered in 1997. The photochemical reactions of semiconductor oxides (including the generation of superhydrophilicity) have received much and widespread attention from environmental and clean energy science. The formation of a very hydrophilic titanium surface by light is caused by a change in the surface structure of the hydrophilic phase formed by light treatment. In this model, light treatment creates a surface oxygen deficiency at the cross-linked site, thereby changing the Ti ⁴⁺ site to a Ti ³⁺ site, which is favorable for dissociative water absorption.

We have 1) newly processed or modified titanium surfaces are positively charged; 2) the old titanium surface is treated with UV radiation so that the surface is positively charged. Treating a newly processed titanium surface with UV radiation enhances its positive charge; 3) These positively charged surfaces exhibit protein and cell affinity and are not treated with UV light There is a substantial increase in protein and cell affinity compared to the surface; 4) These newly discovered and formed mechanisms for protein and cell adhesion are directly between the protein and / or cell and the titanium surface. We have found that this does not require a divalent cation for crosslinking, such as Ca ²⁺ . New surfaces and biological mechanisms can be distinguished from biological processes that have been recognized in the field of titanium implants. This is because protein absorption and cell adhesion, the final titanium surface, is substantially increased in tissue integration and regeneration capabilities.

  The UV ray treatment can be performed under normal air atmosphere conditions, for example, under conditions that do not require other atmosphere settings such as a vacuum state or inert gas injection. When titanium or a titanium mixed metal is treated with UV radiation, it activates electrons from the valence band of titanium atoms to the conductive band, thereby forming positive holes in the surface layer of titanium and forming positive charges on the surface. To do. To activate the electrons, a UV radiation energy of 3.2 eV is required, which corresponds to a wavelength of approximately 365 nm called UVA. On the other hand, in the case of UVC whose peak wavelength is shorter than 260 nm, the hydrocarbon is directly decomposed. This carbon removal mechanism helps the UVA penetrate through the titanium surface, increasing the efficiency of forming a positive charge and facilitating and improving exposing the resulting positive charge.

  Without being limited to any theory, a combination of UVA (about 340 nm to about 380 nm) and UVC (about 170 nm to about 270 nm) was used.

  Improvements in bone-titanium integration mediated by UV-treated titanium have proved substantial. For example, biomedical anchors for acid-etched implants were increased more than 3-fold in the initial recovery of healing at 2 weeks. This 3-fold increase in push-in value was also obtained at 8 weeks in the same animal model. In other words, the push-in value obtained with UV-treated and acid-etched implants is equivalent to the 8th week of implants that have not been UV-treated and etched with acid, ie UV-treated surfaces Indicates that bone-implant integration has been achieved four times faster. UV-promoted titanium makes it possible to achieve an optimal level of bone-titanium direct contact (substantially 100%) without being substantially disturbed by soft tissue. These in vivo achievements were achieved by the following biological processes on UV-treated titanium surfaces: (1) increased protein absorption; (2) increased osteoblast migration; (3) osteoblasts Increased adhesion; (4) Promotion of osteoblast dispersal; (5) Increased proliferation of osteoblasts; (6) Promotion of osteoblast differentiation.

  These processes may or may not be independent. For example, increased protein absorption can improve osteoblast adhesion through the promotion of protein and cellular integrin interactions. Increased osteoblast proliferation can also cause improved differentiation by increasing cell-cell interactions.

  Because the UV-treated surface has increased fibronectin absorption, other cells with RGD adhesion integrins are also attracted to the surface. Interestingly, soft tissue inhibition was substantially reduced around UV-treated titanium.

  In order to form more bone faster, it is necessary to overcome the inverse correlation between osteoblast proliferation and differentiation rate. This is also true when forming bone around the titanium implant. For example, a micro-roughened titanium surface is advantageous over a machined smooth surface, not only because it increases the tissue-titanium mechanical interlock but also osteoblasts. It also promotes cell differentiation, resulting in faster bone formation. However, bone mass is smaller than around the machined surface because osteoblast proliferation is reduced. Surfaces etched with a rougher acid have a relatively reduced cell density and proliferation activity compared to smooth machined surfaces. Substrate materials with a rougher surface generally reduce cell proliferation, where intracellular conditions are associated with slowing or limiting progression of the G1 phase of the cell cycle. there is a possibility. The fact that cell dispersion is promoted on the UV-treated surface may mean that the intracellular state has been eliminated. Cell proliferation is only assessed with a BrdU assay that targets the S phase of the cell cycle.

  Analysis to identify cells in various cycles of the cell cycle, and further analysis of their shape and intracellular state, to see how UV radiation treatment works in regulating osteoblast proliferation To help. It can be seen that the proliferation rate of osteoblasts and the differentiation rate of osteoblasts are increased, as shown in the results of slightly increased ALP activity and gene expression. This allows the UV-treated surface to increase osteoblast proliferation without sacrificing differentiation. This biological advantage is also evident in cell morphometry results showing that the bone mass around the UV-treated surface has increased approximately 2-fold.

The cell adhesion and proliferation and bone-implant adhesion rate enhanced by UV radiation is also shown in the deposition of titanium tetraisoperoxide with heat treatment to make anatase TiO ₂ crystals. In the present invention, it has been clarified that the light-induced biological effect can be obtained on the surface of the titanium bulk without depositing or sintering titanium oxide.

Another notable finding is that the present invention is compared to anatase TiO ₂ crystals where the bone-implant contact of the 24 hour UV-treated implant is 28% and 17% for the untreated UV. There was a significant increase in bone-implant contact. 48 hours of UV radiation treatment increased bone-implant contact by a factor of 2.5 during the initial healing phase of the second week of the study. UV treatment intensity, wavelength and time, as well as differences in the surface chemistry of the titanium used, affected different biological effects. Prior to in vivo studies, it was confirmed that 48 hours of UV radiation treatment was required to generate superhydrophilicity on machined and acid etched surfaces, ranging from 24 to 48 hours. The biological effects increased during the UV treatment, such as cell adhesion ability.

The biological effects generated by light are related to the occurrence of superhydrophilicity, as well as to the reduction in the proportion of atomic carbon, as demonstrated by enhanced and improved protein absorption and cell adhesion. Yes. In order to confirm whether these physicochemical changes are due to the photocatalytic phenomenon of TiO ₂ , the titanium surface used in this study was carefully examined. An absorption band at 300-350 nm is also observed in the titanium sample used, which is typical for TiO ₂ semiconductors. The XPS spectrum showed a 2p _3/2 peak at approximately 458.5 eV, but for 453.8 eV it was not shown in both the machined and acid etched surfaces (FIG. 6D); Ti and TiO ₂ The 2p _3/2 peaks of are known to be 453.8 eV and 458.5 eV, respectively. Furthermore, the shoulder peaks used for reduced species such as Ti ³⁺ and / or Ti ⁰ were not seen in the low energy region on any titanium disk. These data indicate that near the surface of these substrates are fully oxidized to form a thin layer of stoichiometric TiO _{2 and} the carbon fraction that decreases with increasing UV dose is carbonized. This is due to the photocatalytic removal of hydrogen. Furthermore, the 2p _3/2 peak on the surface etched with acid is shifted to a slightly higher energy than the machined surface indicates that the oxide layer is on the surface etched with acid. It indicates a thicker possibility. This can be explained by a more pronounced physicochemical change in the response to UV radiation on the acid etched surface.

Hydrocarbon levels, but not hydrophilic levels, were strongly related to the rate of protein absorption and cell adhesion. From this finding, the amount of hydrocarbons absorbed by TiO ₂ at the time of implantation is important in determining the initial affinity of osteoblasts, and consequently identifies bone morphology in vivo. And is considered important in determining bone-implant integration. On the control titanium surface, the level of protein absorption and the number of cell adhesions are low compared to that of the UV-treated surface and low even after prolonged culture, which is a significant long-term attributed to the initial biological environment. Effects. Titanium implants currently used for medical and research purposes have been identified for hydrocarbon contamination. Accumulation of organic molecules on the titanium surface is considered to be unavoidable in a normal environment, particularly those having a carbonyl moiety. This can be explained for the relatively low bone-titanium contact (45-75%) as described above. The present invention shows that the bone-titanium contact can be increased to nearly 100% by treating the titanium implant with UV radiation.

Tested proteins and osteoblasts are negatively charged. When the oxygen-containing hydrocarbon covering the TiO ₂ surface is removed by UV radiation treatment, Ti ⁴⁺ is exposed. Thereby, interaction between protein and a cell, such a cation etc. can be accelerated | stimulated. The biocompatibility formation of the TiO ₂ surface associated with hydrocarbon photolysis is shown schematically in FIG. 6M.

  In order to minimize failure rates, reduce morbidity, and maximize post-operative functionality, various efforts have been made regarding osseoimplant treatment in both the dental and orthopedic fields. One problem with the placement of implants for rehabilitation is the problem of bones with impaired regenerative capacity and metabolic activity, especially those that slow and further hinder bone-titanium integration. Another problem is that the use of acrylic bone cement in some implant procedures inherently limits the biocompatibility and long-term prediction of the implant. There is a tendency to use cement-free implants to avoid bone-cement complications. From these epidemiological, surgical and social issues, it is well understood that we are trying to develop new implant therapies that are more pluripotent and that can better predict life. The greatest advantage gained from the physicochemical changes of titanium using UV radiation as shown in the present invention is that it is three times stronger than the implant in the initial healing phase, corresponding to four times faster in establishing bone-titanium integration. It is fixed at that time. Since improved osseointegration capabilities have been observed on both machined and acid-etched surfaces, this technology can also be applied to other surface types with the main current titanium implants. You can expect it to be extended and used. Because this technique is simple, efficient, and low cost, it can be used immediately and in a wide range of dental, facial, and orthopedic implant treatments.

  Described herein is a method for imparting functionality to an implant, comprising: (1) providing an implant surface; and (2) treating the implant surface to charge the surface with a positive charge or Enhancing the positive charge on the surface. In some embodiments, the method is positively charged or enhanced under physiological conditions. The physiological condition may be a pH of about 7. In some embodiments, the method causes a positive charge or enhancement even below pH 7 or above pH 7.

For one embodiment, the implant has a titanium surface. In one embodiment, the implant has a metallic or non-metallic carrier material. Titanium surface has a TiO _2. In some embodiments, the treated surface is substantially free of hydrocarbons. In some embodiments, the treated surface has titanium oxide cations.

  The atomic ratio of carbon on the titanium surface can be suppressed to less than 20%, whereas the untreated or old titanium surface is more than 50%.

  The implant surface is treated by applying ultraviolet light (UV radiation). UV radiation can be irradiated using a UV lamp. The UV radiation may have a wavelength of about 10 nm to about 400 nm. In some embodiments, the UV radiation may have a wavelength of about 170 nm to about 270 nm, or may have a wavelength of about 340 nm to about 380 nm. In some embodiments, the surface may be treated with a combination of UV light having a wavelength of about 170 nm to about 270 nm and UV light having a wavelength of about 340 nm to about 380 nm.

The intensity of UV radiation may be in a wide range. For example, the intensity of UV rays, may be between 0.001 mW / cm ² and 100 mW / cm ^2. In some embodiments, the intensity of UV radiation can be about 0.1 mW / cm ² or about 2 mW / cm ^2.

  The treatment with UV light may be within 48 hours, for example, 30 seconds, 1 minute, 5 minutes, 15 minutes, 30 minutes, 1 hour, 5 hours, 10 hours, 24 hours, 36 hours, 48 hours, etc. .

  In one embodiment, the method further includes processing the implant surface prior to treating the implant surface. The implant surface may be physically processed or chemically processed. Physical processing includes machining or sand blasting. Chemical processing includes etching with acid or base. The acid may be sulfuric acid. The newly machined surface may be positively charged. UV radiation treatment enhances the positive charge of the processed surface.

  In one embodiment, the treated surface can attract proteins and cells at an improved rate. As used herein, “improved rate” means that the treated implant surface has a higher rate of attracting cells or proteins compared to the corresponding untreated implant surface. An untreated implant surface includes a newly processed surface, and an “old surface” is, for example, one day, three days, one week, two weeks, three weeks, four weeks, etc. that have been processed since time Say. Increased percentages are 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, 100% , 200%, 300%, 400%, etc., that are higher than the percentage of protein or cells attracted to the corresponding untreated surface.

  As used herein, “improved” may be used in the same way as “improved” or “increased”. Improved means that the numbers are faster, stronger, and higher.

The protein may be bovine serum albumin, fraction V, and bovine plasma fibronectin. The cells may be human mesenchymal stem cells and osteoblasts. Proteins and cells may adhere directly to the treated surface, for example without cross-linking divalent cations and the like. In one embodiment, the treated surface does not include Ca ²⁺ , Mg ²⁺ , Zn ^{2+ and the} like.

  The treated implant surface can improve tissue-implant integration and / or bone-implant integration at the implant site. The treated surface has an improved bone forming ability over the untreated implant surface. The treated surface has a tissue-implant integration, bone-implant integration, or bone forming ability, eg, 5%, 10%, 15%, 20%, 25%, compared to the corresponding untreated surface. 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, etc. it can.

  Treated implant surfaces can: Increase protein absorption, increase osteoblast migration, increase osteoblast adhesion, promote osteoblast dispersion, osteoblasts compared to untreated surfaces Increase cell proliferation and promote osteoblast differentiation. Each activity is, for example, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, 100% , 200%, 300%, 400%, 500%, etc.

Described herein is an implant having a surface imparted with functionality by the method described above. In one embodiment, the medical implant has a titanium surface. The titanium surface has a positively charged TiO ₂ . In one embodiment, the titanium surface is substantially free of hydrocarbons.

  The implant further has a carrier material. In one embodiment, the support material is a metal. In one embodiment, the support material is non-metallic.

  The implant surface can attract proteins or cells at an improved rate. As used herein, “improved rate” refers to a higher rate at which the implant surface attracts cells or proteins than a surface that is not positively charged or has a weak positive charge. The improved percentage is 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45% compared to the corresponding non-positive or weakly charged surface 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, etc.

  The implant surface can attract proteins such as bovine serum albumin, fraction V, and bovine plasma fibronectin. The implant surface can attract cells such as human mesenchymal stem cells and osteoblasts.

  The implant surface allows tissue-implant integration and / or bone-implant integration. The implant surface is 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60 compared to the non-positive or weakly charged surface To improve bone-implant integration, tissue-implant integration, or osteogenic activity at a rate of%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, etc. Can do.

  Implant surfaces can: Increase protein absorption, increase osteoblast migration, increase osteoblast adhesion, osteoblast dispersion compared to non-positive or weakly positive surfaces Promotes, increases osteoblast proliferation, and promotes osteoblast differentiation. Each activity is, for example, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, 100% , 200%, 300%, 400%, 500%, etc.

  Described herein is a novel titanium surface that exhibits improved bioactivity, protein and biological cell adsorption. The titanium surface is positively charged and is formed by treating the new layer of titanium with UV radiation and / or treating the surface with UV radiation. Irradiation of the new titanium layer includes new processing of the surface, such as machining, etching, sand blasting and combinations thereof, and reprocessing of the old surface. The present invention can be used immediately and in a wide range of dental and orthopedic implants, and can also be used in the fields of bone regeneration therapy and bone engineering due to its simplicity, high efficiency and low cost.

  It has been found that UV irradiation of titanium improves osteoconductivity. Is responsible for the effects of UV irradiation on titanium in various in vitro behaviors and functions of osteoblasts on titanium substrates, and the potential for bone-titanium integration in vivo and improved osteoconductivity The factors of the UV-treated titanium surface were examined.

  Described herein is a method for improving the osteoconductivity of titanium and a titanium surface with improved osteoconductivity using the method. Machined titanium samples and acid etched titanium samples were treated with UV radiation at various times within 48 hours. For both surfaces, UV radiation treatment increased the rate of adhesion, dispersion, proliferation and differentiation of rat bone marrow-derived osteoblasts, and also increased protein absorption capacity by a factor of three. In vivo cell morphometry in a rat model reveals that UV-treated implants have a wide range of bone formation without being disturbed by soft tissue, which is the healing phase at 4 weeks. The bone-implant contact is maximized to near 100%.

Implant biomechanical testing revealed that UV radiation treatment accelerated implant fixation four times. Proportion of protein absorption and cell adhesion has been strongly associated with UV dose corresponding atomic ratio of carbon in the TiO _2, it was not associated with the state of the hydrocarbon. Pretreatment of titanium with UV radiation, in conjunction with UV-catalyzed progressive removal of hydrocarbons from the TiO ₂ surface, has substantially improved osteoconductive ability, which is faster and more complete It suggests functionalization of titanium with light that enables bone-implant integration.

UV radiation treatment of the titanium surface significantly increased the osteoconductive ability of the titanium surface. New bone formation occurs extensively in UV-treated implants without substantial soft tissue interference, although this maximizes bone-implant contact by nearly 100% at the 4 week healing stage. On the contrary, for untreated implants, bone-implant contact was about 50%. The UV radiation treatment increased the strength of the bone-titanium integration three times during the healing phase at 2 weeks. The UV-treated surface created an osteoblast-friendly environment, as shown by improved osteoblast adhesion, dispersion, proliferation and differentiation, as well as increased protein absorption. Proportion of protein absorption and cell adhesion has been strongly associated with UV dose corresponding atomic ratio of carbon in the TiO _2, it was not associated with the state of the hydrocarbon. This titanium bioactive UV-mediated enhancement was also demonstrated on differently shaped surfaces, machined implant surfaces and acid etched surfaces. Therefore, here is provided a method for imparting optical functionality of titanium that allows faster and perfect bone-titanium integration.

Medical implants Medical implants may be metallic or non-metallic. In some embodiments, the medical implant is a metal implant, such as a titanium implant, such as one that replaces a missing tooth (dental implant) or a titanium implant used for disease, fracture or transplanted bone. is there. Examples of other metal implants include titanium alloy implants, chromium-cobalt alloy implants, platinum and platinum alloy implants, nickel and nickel alloy implants, stainless steel implants, zirconium, chromium-cobalt alloys, gold or gold alloy implants and aluminum or aluminum Including but not limited to alloy implants.

  The metal implants described herein include titanium implants and non-titanium implants. Titanium implants include tooth or bone substitutes made from titanium and titanium-containing alloys. Titanium bone substitutes include, for example, knee and hip prostheses, femoral neck substitutes, spine substitutes and repairs, neck bone substitutes and repairs, jawbone repairs, fixations and augmentations, transplants Includes bone fixation and other limb prostheses. Non-titanium metal implants include gold, platinum, tantalum, niobium, nickel, iron, chromium, titanium, titanium alloys, titanium oxide, cobalt, zirconium, zirconium oxide, magnesium, aluminum, palladium, and, for example, stainless steel and combinations thereof Tooth or bone implants formed from these alloys and the like. Examples of other alloys include titanium-nickel alloy, nitinol, chromium-cobalt alloy, stainless steel, or combinations thereof. In some embodiments, the metal implant can specifically exclude the metals described above.

  Non-metallic implants include, for example, ceramic implants, calcium phosphate or polymer implants. The polymer implant that can be used may be any biocompatible implant, such as a biodegradable polymer implant. Examples of the ceramic implant include bioglass and silicon dioxide glass. The calcium phosphate glass includes, for example, hydroxyapatite, tricalcium phosphate (TCP), and the like. Examples of polymer implants include, for example, polylactic acid / glycolic acid (PLGA), polyacrylates such as polymethacrylates and polyacrylates, and polylactic acid (PLA) implants.

  In some embodiments, the implant comprises a metal implant and a bone-cement material. The bone cement material may be any bone cement material known in the art. Some examples of bone cement materials include polyacrylates, polymethacrylate materials such as polymethyl methacrylate (PMMA) / methyl methacrylate (MMA), polyester materials such as PLA or PLGA, bioglass, ceramics , Calcium phosphate-based materials, calcium-based materials, and combinations thereof, but are not limited thereto. In some embodiments, the medical implant may be any polymer described below. In some embodiments, the aforementioned materials for medical implants may be specifically excluded.

  “Osteogenic ability” or “osteoconductivity” refers to the ability to form bone. It also refers to the improved ability of bone integration for medical implants by implants. Bone integration ability refers to the ability of a medical implant to integrate into a living bone. The tissue integration ability refers to the ability of the medical implant to integrate into the living tissue.

UV-irradiation referred to herein as "irradiating UV radiation", "photoactivatable", "light radiation", "light irradiation", "UV radiation activation", "UV radiation" or "UV-irradiation Can be used in the same way. Radiation having a wavelength of 400 nm to 10 nm is generally called UV radiation.

  The medical implant may be emitted with or without sterilization. Anyone with ordinary technical knowledge can sterilize medical implants during UV radiation.

  In one concept of the present invention, a facility or apparatus for irradiating a medical implant has been provided. In one embodiment, the facility or apparatus includes a chamber in which a medical implant is placed, a light source of high energy radiation, and a switch that turns radiation on or off. The facility or device may further include a timer. In some embodiments, the facility or apparatus further includes a mechanism that rotates or spins the medical implant or UV radiation source to completely irradiate the implant. Alternatively, the chamber in which the medical implant is placed may have a reflective surface that allows it to radiate from a different angle, for example any angle of 360 degrees. In some embodiments, the facility or device may include mechanisms that preserve enhanced bone integration capabilities, such as multiple exposures of light, radiolucent implant packages, packing, and ships.

Medical Applications The medical implants described herein can be used to treat, prevent, ameliorate, or alleviate medical symptoms resulting from implanting a medical implant in a mammalian subject. The mammalian subject may be a human or a vertebrate such as a dog, cat, horse, cow, bull or monkey.

  Typical examples of medical conditions that can be treated or prevented with the implants provided here are missing teeth or bone-related symptoms such as femoral neck fractures, missing teeth, femoral neck fractures, neck bone fractures, wrist joints Caused by disorders or symptoms such as fractures, vertebral fractures / disorders or joint fractures and degenerative changes such as intervertebral disc herniation, rheumatoid knee, eg cancer, injury, systemic metabolic disorders, infection or aging and combinations thereof Includes those requiring orthopedic fixation, such as bone or other tissue abnormalities or defects, and bone-related symptoms.

  In some embodiments, the medical implant provided herein is a missing tooth or a bone-related condition such as a femoral neck fracture, a missing tooth, a femoral neck fracture, a neck bone fracture, a wrist joint fracture, a vertebral fracture / Bones caused by disorders or symptoms and combinations of disorders and symptoms such as cancer, injury, systemic metabolic disorders, infections, aging and amputation, etc., and fractures and degenerative changes in joints such as disc herniation, rheumatoid knee It can be used to treat, prevent, ameliorate, or alleviate medical conditions such as those requiring orthopedic fixation such as other tissue abnormalities or defects and bone related symptoms.

  Although specific embodiments of the present invention have been described, various changes and modifications can be made without departing from the technical scope of the present invention. Accordingly, the appended claims are intended to cover these modifications and variations within the scope and spirit of this invention.

For titanium samples, surface analysis and UV-treated cylindrical implants (diameter 1 mm, length 2 mm) and discs (diameter 20 mm, thickness 1.5 mm), two surfaces were prepared with commercially pure titanium. One had a machined surface that was rotated on the wheel and the other was 67% H ₂ SO ₄ etched with acid at 120 ° C. for 75 seconds. In addition, a machined surface and a sandblasted surface were prepared. All surfaces were analyzed for optical properties and spectrophotometers (UV-2200A, Shimadzu, Tokyo, Japan) and X-ray diffraction (XRD) (XRD-6100, Shimadzu, Tokyo, Japan), respectively. The crystal structure was examined. The hydrophilic state of the titanium surface was examined using a contact angle meter (CA-X, Kyowa Interface Science, Tokyo, Japan). All work was done in a Class 10 clean room in an environment controlled at 20 ° C. and 46% humidity.

The chemical composition of the titanium surface was evaluated by photoelectron spectroscopy (ESCA). ESCA was performed using high pressure reduction (6 × 10 ⁻⁷ Pa) X-ray photoelectron spectroscopy (XPS) (ESCA 3200, manufactured by Shimadzu Corporation, Tokyo, Japan). Titanium discs and cylindrical implants treated with UV radiation for various times up to 48 hours were compared to untreated controls for surface properties and biological potential. UV radiation treatment was performed using a 15 W germicidal lamp (Toshiba, Tokyo, Japan); strength; ca. It was performed using 0.1 mW / cm ² (UVA: λ = 360 ± 20 nm) and ² mW / cm ² (UVC: λ = 250 ± 20 nm).

  The active ability of the titanium surface was also tested using UVA and UVC separately.

  The UV ray treatment can be performed in a normal air state without any atmosphere setting such as reduced pressure or inert gas injection. When the titanium or titanium-containing metal is treated with UV radiation, the electrons in the valence band of the titanium atom are raised to the transmission band, thereby forming a positive hole in the surface layer of titanium and causing the surface to be positively charged. In order to raise this electron, UV radiation energy of 3.2 eV is necessary, which corresponds to a wavelength of 365 nm and is called UVA. In contrast, the direct decomposition of hydrocarbons is performed by UVC, and this peak wavelength is 260 nm. This carbon removal facilitates the passage of UVA through the titanium surface, increases the efficiency of positive charge formation and accelerates the exposure of the resulting positive charge.

  Without being limited to any theory, a combination of UVA (about 340 nm to about 380 nm) and UVC (about 170 nm to about 270 nm) was used.

Measurement of protein absorption Bovine serum albumin, fraction V (Pierce Biotechnology, Inc, Rockford, Ill.) And bovine plasma fibronectin (Sigma-Aldrich, St. Louis, MO) were used as model proteins. 300 ml of protein solution (1 mg / ml protein / saline) was spread on a Ti disk using a pipette. After culturing for several different times at 37 ° C. in sterilized and humidified conditions (eg, culturing for 2, 6, 24, or 72 hours), the non-adhesive protein is removed and saline containing 0.9% sodium chloride is added. Washed twice using. The initial culture (200 μl) and the removed solution were mixed with 200 μl of microbicinchoninic acid (Pierce Biotechnology, Inc, Rockford, Ill.) And incubated at 37 ° C. for 60 minutes. Protein quantification was performed using a microplate reader at 562 nm.

Human Mesenchymal Stem Cells Human mesenchymal stem cells (MSCs) (Poietics cells, Cambrex Bio Science, Walkersville, East Rutherford, NJ) were cultured in MSC growth medium consisting of MSC basal medium and growth supplements (SingleQuots). Growth supplements include fetal bovine serum (FBS), L-glutamine and penicillin / streptomycin. The cells were cultured in a humidified atmosphere, 95% air, 5% CO ₂ and 37 ° C. In the final passage at 80% cell saturation, cells were detached using 0.25% trypsin-1 mM EDTA-4Na and seeded on a Ti disk at a density of 3 × 10 ⁴ cells / cm ² . The medium was renewed every 3 days.

Bone marrow cells isolated from the femurs of osteoblast cultured Sprague-Dawley rats were treated with 15% fetal calf serum, 50 μg / ml ascorbic acid, 10 mM Na-β-glycerophosphate, 10 ⁻⁸ M dexamethasone, and Placed in alpha-modified Eagle's medium supplemented with antibiotic-antifungal solution. Cells were cultured at 37 ° C. in humidified 95% air, 5% CO ₂ . At 80% cell saturation, the cells were detached using 0.25% trypsin-1 mM EDTA-4Na, and UV-irradiated on a titanium disk etched or acid etched at a cell density of 3 × 10 ⁴ cells / cm ² . Treatment and UV treatment were applied. The culture medium was renewed every 3 days.

Migration assay Migration of human MSCs to the titanium surface was tested using a dual chamber migration assay (345-024K, Trevigen, Gettysburg, Md.). Cells were placed in the upper chamber. The Ti disk was placed at the bottom of the lower chamber. After culturing at 37 ° C. for 3 hours, the percentage of cells that passed through a polyester membrane having a diameter of 8 μm and moved to the lower chamber was stained with calcein-AM and then analyzed with a plate reader.

Cell Adhesion, Density, Proliferation Assay Initial cell adhesion was assessed by measuring the number of cells attached to the titanium surface after 3 and 24 hours of culture. In addition, proliferated cells were quantified as cell density on days 2 and 5 of culture. These quantifications were performed using a WST-1 spectrophotometric analyzer (WST-1, Roche Applied Science, Mannheim, Germany). Medium wells were incubated with 100 μl tetrazolium salt (WST-1) reagent for 4 hours at 37 ° C. The formazan product was measured using an ELISA reader at 420 nm. Furthermore, the cells were stained with calcein-AM and observed under a fluorescence microscope to confirm the cell density.

  Cell proliferation activity was measured by the amount of BrdU incorporation during DNA synthesis. On the second day of culture, 100 μl of a 100 mM BrdU solution (manufactured by Roche Applied Science, Mannheim, Germany) was added to the medium well and cultured for 10 hours. After trypsinization of the cells and DNA denaturation, the medium was incubated with anti-BrdU conjugated with peroxidase for 90 minutes and reacted with tetramethylbenzine for color development. Absorption at 370 nm was measured using an ELISA reader (Synergy HT, BioTek Instruments, Winnocky, VT).

Cell morphology and morphometric confocal laser scanning microscope was used to confirm the morphology and cytoskeletal structure of human MSCs. Three hours after culturing, the cells were fixed in 10% formalin, and further stained with a fluorescent dye, rhodamine phalloidin (actin filament was red, Molecular Probe, OR). The medium was stained with a mouse anti-paxillin monoclonal antibody (Abcam, Cambridge, Mass.) And a FITC-conjugated anti-mouse secondary antibody (Abcam, Cambridge, Mass.) Was added. Cell area, perimeter, and ferret diameter were quantified and analyzed with an image analyzer (ImageJ, NIH, Bethesda, MD).

After 3 hours of culture, osteoblasts were fixed with 10% formalin and stained with fluorescent dyes, DAPI (cell nucleus blue, Vector, CA) and rhodamine phalloidin (actin filament red, MicroProbe, OR). A confocal laser scanning microscope was used to confirm cell morphology and cytoskeletal structure. Cell area, perimeter, and ferret diameter were quantified and analyzed with an image analyzer (ImageJ, NIH, Bethesda, MD).

Alkaline phosphotase (ALP) activity ALP activity of cultured osteoblasts was confirmed by medium area and spectrophotometric assay. The cultured osteoblasts were washed twice with Hank's solution and 30 minutes at 37 ° C. with 120 mM Tris buffer (pH 8.4) containing 0.9 mM naphthol AS-MX phosphate and 1.8 mM Fast Red TR. Cultured. ALP positive areas in stained images were analyzed using image analysis software (Image Pro-plus, Media Cybernetics, Silver Springs, MD, USA) as [(stained area / total incubator area) × 100] (%). calculated. For spectrophotometric analysis, the medium was washed with ddH ₂ O, added with 250 μl of p-nitrophenyl phosphate (LabAssay ATP, Wako Pure Chemical Industries, Ltd., Richmond, VA) and incubated at 3TC for 15 minutes. did. ALP activity was assessed by the amount of nitrophenol released via the enzymatic reaction and measured at a wavelength of 405 nm using an ELISA reader (Synergy HT, BioTek Instruments, Winnoseki, VT).

Mineralization Assay The mineralization ability of cultured osteoblasts was confirmed using a spectrophotometric assay on the mineralized mass area. The mineralized mass of osteoblasts was visualized by von Kossa staining. The culture was fixed with 50% ethanol / 18% formaldehyde solution for 30 minutes. The culture was incubated with 5% silver nitrate for 30 minutes under UV light. The culture was washed twice with ddH ₂ O and cultured for 2-5 with 5% sodium thiosulfate solution. The mineralized mass area is calculated as [(stained area / total incubator area) x 100] (%) using image analysis software (Image Pro-plus, Media Cybernetics, Silver Springs, MD, USA). It was. For spectrophotometric detection of calcium deposition, the culture was washed with PBS and cultured overnight with gentle shaking in 1 ml of 0.5 M HCl solution. The solution was mixed with ortho-cresolphthalein complexone in an alkaline solvent (calcium binding and buffering reagent, Sigma, St. Louis, MO) to form a red calcium-cresolphthalein complexone complex. The color intensity was measured by measuring absorbance at 575 nm using an ELISA reader (Synergy HT, BioTek Instruments, Winnoseki, VT).

Gene expression analysis Gene expression was semi-quantified using reverse transcription polymerase chain reaction (RT-PCR). Total RNA was extracted from the cultures using Trizol (Invitrogen, Carlsbad, CA) and a purification column (RNeasy, Qiagen, Valencia, CA). After DNAseI treatment, reverse transcription of 0.5 μg of total RNA using MMLV reverse transcriptase (Clontech, Carlsbad, CA) in the presence of oligo (dT) primer (Clontech, Carlsbad, CA) went. The PCR reaction was performed using Taq DNA polymerase (EX Taq, Takara Bio, Madison, WA), and collagen I, osteopontin and osteocalcin mRNA were detected using the primer design and the PCR conditions described above. PCR products were visualized on 1.5% agarose gels stained with ethylene bromide. Band intensities were quantified under UV radiation and normalized to GAPDH mRNA.

Male Sprague-Dawley rats 8 weeks after surgery were anesthetized with 1-2% isoflurane aspiration. After shaving the foot and scrubbing with 10% Providone-Iodine solution, the distal region was carefully exposed through a skin incision and muscle cut in the femur. As the distal region of the femur, a flat surface was selected for implant placement. The implant site was prepared 9 mm from the distal tip of the femur using a 0.8 mm round bar and expanded using a reamer (#ISO 090 and 100). A large amount of irrigation with sterile isotonic salt solution was used for cooling and washing. One cylindrical implant was placed on each side of the femur. The surgical site was then closed in layers. Muscle and skin were sewn separately with resorbable thread. The protocol has been certified by the chair of the Animal Experiment Committee at the University of California, Los Angeles (UCLA), and all experiments were conducted in accordance with the United States Department of Agriculture Animal Experiment Guidelines.

Implant Biomechanical Push-In Test The implant biomechanical push-in test was used to assess the biomechanical strength of the bone-implant integration. The femur containing the cylindrical implant was harvested and embedded in a self-polymerizing resin with the implant level top surface. MicroCT was used to verify that the implant was not receiving bone cortical support from the side and bottom of the implant. Using a testing machine (Instron 5544 electromechanical universal testing machine, Instron, Canton, Mass.) With a 2000N load cell and a pushing rod (diameter = 0.8 mm), at a tip speed of 1 mm / min, perpendicular to the implant The load was applied downward. The push-in test was decided to measure the peak of the load displacement curve.

Femurs with implants etched with cell preparation acid were harvested and fixed in 10% buffered formalin at 4 ° C. for 2 weeks. The specimens were dehydrated by stepwise alcohol cleaning and embedded in a photocured epoxy resin (Technobit 7200 VLC, Heraeus Kultzer, Welheim, Germany) without decalcification. The embedded specimen was sawed perpendicular to the long axis of the cylindrical implant at 0.5 mm from the tip of the implant. The specimens were ground to a thickness of 30 μm using a grinding system (ExakApparatebau, Norderstedt, Germany). The ground pieces were stained with Goldner trichrome stain and observed with an optical microscope.

Cell Morphology Using a 40 × lens and a 4 × zoom computer screen, images were used for computer cell morphometry (Image Pro-plus, Media Cybernetics, Silver Springs, MD). In order to identify the cell structure in detail, an electron microscope with a maximum magnification of 400 was used. The distinction between implant-related bones and non-implant-related bones has already been mentioned. According to this method, the tissue surrounding the implant was divided into two zones as described below: (i) a proximal region within 50 μm from the implant surface and a peripheral edge (ii) distal within 50 μm to 200 μm from the implant surface Region and perimeter. The following variables were analyzed:
Bone-implant contact rate (%) = (total bone-implant contact length) / (implant circumference) × 100, where bone-implant contact is within 20 μm from the implant surface without being disturbed by soft tissue The interface with the bone tissue in the area.
Bone mass in proximal region (%) = (bone area in proximal region) / (area of proximal region) × 100
Bone mass in distal region (%) = (bone area in distal region) / (area of distal region) × 100
Soft tissue interference (%) = (total length of soft tissue interfering between bone and implant) / (total length of bone surrounding the implant) × 100

Statistical analysis Three samples were used for cell culture observation except for cell morphometry which required 10 cell samples. Two-way ANOVA was used to confirm the effect of the presence or absence of UV irradiation on the titanium surface of an age different from the culture time. If necessary, a post hoc Bonferoni test may be performed to examine the difference between the new machined surface, the 4th week surface, and the 4th week surface treated with UV radiation. There is a significant difference. If the data was only at one time point, one-way ANOVA was used to confirm the test group differences. A T-test was also conducted to examine the difference between untreated controls and UV treated groups. The correlation between albumin absorption and cell adhesion, carbon atomic ratio and H ₂ O contact angle was examined, and a regression curve equation was obtained by least mean square approximation.

Result 1. Accelerated and improved protein absorption on newly processed and UV-treated titanium surfaces Two-way ANOVA showed that albumin absorption was significantly different between test groups (p-value <0 .01; FIG. 1A): freshly etched surface (immediately after processing), 4 week surface (ie, stored for 4 weeks), UV treated 4 week surface. After 2 hours of culture, only about 10% of the cultured albumin was absorbed on the titanium surface at 4 weeks, but about 60% of albumin was absorbed on the newly processed surface ( p value <0.01; Bonferoni method). The amount of albumin absorption was 40% or less on the 4 week surface even after 72 hours of culture compared to the newly processed surface (p value <0.01). The UV-treated surface at 4 weeks showed albumin absorption equivalent to the freshly processed surface after 2 and 24 hours of culture, and a better level after 72 hours (p value < 0.05).

2. Improved stem cell migration and adhesion on freshly processed and UV-treated titanium surfaces The number of human mesenchymal stem cells (MSCs) that migrated through the 8 μm pore varies significantly depending on the culture conditions (P value <0.01, 1-way ANOVA; FIG. 1B). The number of cells that migrated to the 4 week surface after 3 hours of culture was 50% of the number observed on the freshly processed surface, the number observed on the 4 week surface treated with UV radiation. 25% (p value <0.01). The UV treated 4 week surface showed twice as much cell migration as the newly processed surface (p value <0.01).

  The number of human MSCs cells attached to the Ti surface increased in the following order: UV treated 4 week surface> newly processed surface> 4 week surface (p value <0.01; 2-way ANOVA; FIG. 1C). The number of cells adhered to the surface at 4 weeks was less than 50% of the newly processed surface. The UV treated 4 week surface showed substantially higher cell adhesion after 24 hours than the freshly processed surface (> 120%) (p value <0.01).

3. Accelerated cell dispersal and cytoskeletal development of freshly processed surfaces and UV-treated Ti surfaces. Low-magnification images after 3 hours of incubation of actin filament-stained (rhodamine phalloidin) human MSCs, 4 weeks after UV treatment It shows the highest number of cells on the surface of the eye and the lowest on the surface of the 4th week, which is the same as the result of the cell adhesion assay (FIG. 2A). In the high magnification image stained with actin, the newly processed surface and the cells on the surface of the 4th week treated with UV rays have clearly large protrusions extending in several directions, whereas the cells on the surface of the 4th week are Until now, the development of the cytoskeleton has been little. It was observed that in the newly engineered surface and UV treated 4 week surface cells, paxillin, a protein that regulates cell adhesion and adhesion, was strongly localized along the cell shape. In particular, the cells on the surface of the 4th week treated with UV radiation showed a strong positive staining of cell protoplasts.

  Cell morphometry evaluation area, margin, and ferret diameter showed significantly significant differences for these parameters in the three Ti substrates (ANOVA, p-value <0.01; FIG. 2B). These parameters were 5-8 times greater on the newly processed and UV treated surfaces compared to the 4 week surface (Bonferoni method; p value <0.01). There was no noticeable difference between the newly processed surface and the UV treated surface.

4). Improved in vivo bone-titanium integration on the newly machined surface and the UV-treated Ti surface In vivo implant fixation is the most important factor for the medical capabilities of titanium implants as load bearing devices. The in vivo stability of the titanium implant was examined by a biomechanical push-in test in a rat model. A cylindrical implant was placed in the rat's femur. The strength of the bone-titanium integration measured by the push-in test in the rat in vivo model is higher for the newly processed and UV-treated surfaces compared to the 4th week surface. In the healing stage, they were 2.8 times and 3.1 times, respectively.

  FIG. 3 shows improved bone-titanium integration with the newly processed and UV-treated surfaces compared to the 4 week surface, as assessed by a biomechanical push-in test.

5). Protein attraction on positively charged surfaces on freshly processed and UV-treated titanium surfaces FIG. 4A shows albumin absorption for various titanium surfaces with different media pH conditions. Absorption on the untreated 4-week titanium surface at pH 7 was limited to 10-15%. The results were predictable because the titanium surface and albumin that are normally available are both negatively charged at biological pH, which inhibits the titanium-albumin interaction. Absorption of albumin increased only when the 4 week surface was treated with a divalent cation such as CaCl ₂ prior to albumin absorption. This can be explained by the fact that the divalent cation deposited on the titanium surface, which is a monovalent anion, serves as a bridge between the negative albumin molecule and the titanium surface.

On the other hand, as described above, the newly processed and UV-treated surfaces show higher absorption rates compared to the untreated surfaces, which are> 35% or> 55% respectively at pH 7. (P value <0.01; untreated group, FIG. 4A). However, in the medium prepared at pH 3, protein absorption on these surfaces was as low as that in the untreated 4th week. This is because the equipotential pH of albumin is 4.7 to 4.9, and albumin undergoes a neutral-basic transition, and at a low value such as pH 3, is positively charged. On the other hand, albumin undergoes a neutral-acidic transition at a high pH value such as pH 7, and is negatively charged. These results show that the newly processed surface and the UV-treated surface are positively charged and further have the property of attracting different proteins depending on the pH value. Furthermore, treating these surfaces with monovalent anions such as NaCl and CaCl ₂ solutions and neutralizing the positive charge on these surfaces, compared to the baseline of the untreated 4 week surface, These albumin absorptions did not increase, confirming the positive charge properties of these surfaces.

  Newly processed and UV-treated surfaces can maintain a positive charge after these ion treatments, and can maintain a low level of surface carbon even at pH 3. These results indicate that the surface positive charge mainly regulates the bioactivity of the titanium surface such as protein absorption, superhydrophilic effects and carbon level defeat.

6). Cell adsorption on positively charged surfaces of freshly processed and UV-treated titanium FIG. 4B shows the number of human mesenchymal stem cells (MSCs) attached to various titanium surfaces prepared in the same manner as FIG. 4A. Indicates.

This experiment was performed at biological pH 7. Since both the titanium surface and the repulsive force of the cells are negatively charged, the number of cells adhering to the surface at 4 weeks was expected to be limited. Conversely, it was expected that there were more adherent cells on the newly processed and UV treated surfaces compared to the 4 week surface. The number of adhered to the newly machined surface and UV-ray treated surface cells, Cl ^- is treated with an anion such as, decreased to baseline untreated 4 weeks of surface. In view of what is already known, biological cells are negatively charged, and the newly processed and UV-treated surfaces are positively charged, so cell-titanium Integration improved.

  Newly processed and UV-treated titanium surfaces can maintain a negative charge after these ion treatments, and can maintain surface carbon at low levels even at pH 3. These results indicate that the surface positive charge mainly regulates the bioactivity of the titanium surface such as protein absorption, superhydrophilic effects and carbon level defeat.

The mechanism of protein and cell adhesion to the titanium surface is described in the figure (FIG. 5). The figure on the left (old titanium) shows the mechanism that occurs around the titanium surface. In this mechanism, cell adhesion must be cross-linked to a divalent cation such as Ca ²⁺ in order to absorb negative proteins and subsequently absorb cells via the protein's RGD sequence. Also, competitive binding of monovalent cations such as Na ⁺ and K ⁺ blocks the anion sites on the titanium surface for Ca ²⁺ binding. As a result, the number of cells that can bind to the titanium surface is reduced.

  The figure on the right (new or UV-treated titanium) represents a novel mechanism based on the present test results, which is a titanium surface that has changed from cell repulsion to cell affinity. Electrostatic positive charges on the newly processed and UV-treated surfaces allow negatively charged proteins and cells to adhere directly to the titanium surface without the help of divalent cations, resulting in adhesion to the surface The number of cells to increase.

7). Protein generalization and cytophilicity of newly processed and UV-treated titanium surfaces In addition to acid etched titanium surfaces, machined and sandblasted titanium surfaces were tested and newly processed. The possible advantages of the treated and UV-treated surfaces were verified (FIG. 6A). The surface at 4 weeks showed 20-45% albumin absorption compared to the newly prepared surface. The UV treatment of the titanium surface at 4 weeks increased to a similar absorption rate to the newly machined surface by machining or was higher than the newly machined surface by sandblasting.

  A similar trend was observed in the proportion of fibronectin absorption (FIG. 6B). Absorption was tested for all surface morphology and was higher in the order of UV treated 4 week Ti, freshly processed Ti, and 4 week Ti (p value <0.01).

  The in vivo achievement of bone-titanium integration was tested using machined titanium. On the machined surface treated with UV radiation, the strength of the bone-titanium integration was significantly increased in the healing phase at 2 and 4 weeks (p value <0.05; FIG. 6C).

  These results indicate that the biological advantages of the newly processed titanium surface and the UV-treated surface are common to different surface treatments and even effective on different proteins.

8). Photoinduced superhydrophilicity of titanium After 48 hours of UV irradiation on titanium discs, the contact angles of the H ₂ O drops were 53.5 ° and 88.4 ° on the machined and acid etched surfaces, respectively. But it was down to 0 °. This suggests a change from a hydrophobic surface to a superhydrophilic surface (FIG. 7A). Superhydrophilicity was formed even faster on surfaces etched with acid. The acid-etched surface required 1 hour of UV irradiation, whereas the machined surface required 48 hours (FIG. 7A). After 48 hours of UV irradiation, the superhydrophilic state lasted longer on the acid-etched surface and further persisted for 7 days in the dark with the H ₂ O droplet contact angle at 0 ° (FIG. 7B). In contrast, on the machined surface, superhydrophilicity began to disappear soon.

9. Enhanced protein absorption by titanium UV radiation In both surface types (machined surface and acid etched surface), UV radiation treatment promoted absorption of albumin and fibronectin (FIGS. 7C and 7D). For example, the albumin absorption rate was <10% after 2 hours of culture, but increased to 50-60% on the titanium surface after 48 hours of UV irradiation (p value < 0.01) (FIG. 7C). For both proteins, the UV-treated effect was greater on the acid etched surface than on the machined surface (p value <0.01). The amount of these proteins on the untreated surface is lower than the UV-treated surface even after 24 hours of culture, indicating that UV treatment promotes and increases protein absorption by almost 100%. (FIGS. 7C and D).

10. Improved osteoblast adhesion in UV-irradiated titanium After 3 hours of incubation, the number of cells adhered on the UV-irradiated surface, both on the machined surface and the acid-etched surface, is untreated. There were 3 to 5 times more compared to the control surface (FIG. 7E). The effect of cell adhesion induced by UV radiation was as high as 24 hours.

11. Biological effects dependent on UV dose To confirm UV-stimulated protein absorption and osteoblast adhesion, UV dose-dependent protein absorption and osteoblast adhesion were tested. Acid etched titanium surfaces were treated with UV radiation for various different times up to 48 hours. UV dose had different effects on protein and cell adhesion (FIGS. 7F and 7G). The increase in albumin absorption occurred quickly and then saturated with 1 hour UV treatment. Cell adhesion rate continued to increase significantly with 48 hours of UV irradiation (p value <0.01).

12 Improved proliferation and accelerated dispersal of osteoblasts with UV-treated titanium Dispersion of osteoblasts and cytoskeletal development on the machined control surface follows a trace 3 hours after machining Isotropic. In these cells, cell processes did not develop well. In contrast, cells on the machined surface treated with UV radiation developed lateral foot-like cell protrusions in multiple directions (image in FIG. 8A). The surface etched with the UV-treated acid had clearly larger cells and longer cell protrusions than the surface etched with the untreated acid. Cell morphometry evaluation of cell area, perimeter, and ferret diameter showed better results for these parameters on the UV-treated titanium surface (histogram in FIG. 8A).

  The cell density is continually greater on the UV-treated surface compared to the UV-untreated machined surface and acid-etched surface after 2 and 5 days in culture (histogram in FIG. 8B), This is consistent with the result of fluorescence image of cells after calcein staining (upper figure in FIG. 8B). In day 2 culture, cellular BrdU incorporation was higher on the UV-treated surface, indicating increased osteoblast proliferation (FIG. 8C).

13. Improved osteoblast phenotype on UV-treated titanium On day 10, ALP-positive areas were observed on UV-treated and acid-etched surfaces compared to control surfaces. It was 2 times or more (the upper and lower histograms in FIG. 9A). In addition, ALP activity, optically quantified and normalized by cell number, was significantly higher on the UV-treated surface (lower right histogram in FIG. 9A).

  At 14 and 28 days in culture, the area of mineralized mass detected by von Kossa staining was also larger on the UV-treated titanium surface; this is more pronounced on the acid etched surface Yes, it showed a 120% increase on day 14 (upper image and lower left histogram in FIG. 9B). The total amount of calcium deposited was consistent with the results of von Kossa staining (lower right histogram in FIG. 9B). RT-PCR analysis has shown similar results with respect to collagen I, osteopontin, and osteocalcin expression throughout the entire culture period, with or without UV irradiation. The expression level increased to <30% on the line-treated surface (FIGS. 9C and D).

14 In Vivo Implant Immobilization Enhanced by UV Radiation The strength of bone-titanium integration measured by push-in test was etched by UV-treated machined surface and acid in the initial healing phase at 2 weeks It was 1.8 times and 3.1 times on the surface, respectively (FIG. 10). In the late healing phase (week 4), the intensity of the osseointegration of the UV-treated implants is 50% on the machined and acid-etched surfaces, respectively, for the UV-untreated implants and 60% better.

15. Bone morphology around UV-treated implants In the second week, the formation of immature bone tissue as woven relatively far from the implant surface in both the control and UV-treated acid-etched implants. Was seen (FIGS. 11A and 11B). Observation of adjacent areas of the implant surface revealed differences in bone morphology between the two implants. Bone formation occurred more significantly around the UV-treated implants (FIGS. 11E and 11F). Another notable difference is the extent of interference by soft tissue. The bone tissue around the untreated control implant had soft tissue between the bone and the implant (FIG. 11I), which was less observed around the UV-treated implant (FIG. 11J). In some sites of the untreated control at 4 weeks, inhibition by fibrous connective tissue was observed between the bone and the implant (FIGS. 11C, G, K), whereas UV treated implants Bone was directly deposited (FIGS. 11D, 11H, 11L).

  In bone cell morphology, the bone-implant contact rate of implants etched with UV-treated acid was always greater than that of the control (2.5 times at 2 weeks and 1.9 at 4 weeks). (Times) (Figure) 11M). The percentage of bone-implant contact area was 98.2% on the UV treated surface. Bone mass in the proximal region of the implant surface was always greater for UV-treated implants than for control implants (FIG. 11N). In contrast, in the distal region, there was no difference in bone mass due to UV rays, indicating that bone formation improved by UV rays is specific to the site adjacent to the implant surface (FIG. 110). A significant decrease in inhibition by soft tissue was confirmed by UV treatment (FIG. 11P). The UV-treated surface completely blocked soft tissue inhibition at the bone-implant interface at 4 weeks, and> 20% of untreated surface bone was between 2 and 4 weeks. There was soft tissue inhibition at the titanium interface.

16. Inverse correlation between carbon atoms on titanium and osteoblast and protein adsorption XRD analysis showed no peaks at 25 ° and 28 ° on both machined and acid etched surfaces These peaks are typical of anatase and rutile TiO ₂ crystals. These surfaces only showed a diffraction pattern derived from Ti metal. However, the UV-VIS absorption spectra of both titanium disks showed an absorption band between 300 and 350 nm (FIG. 12B). The absorption edge of the acid etched surface was in a slightly longer wavelength than the machined surface.

X-ray photoelectron spectroscopy (XPS) spectra showed Ti2p, OIs, and Cls peaks on both titanium surfaces, but no other peaks, indicating that contamination of impurities other than these elements The narrow spectrum of Ti2p shows no clear peak at 2p _3/2 at about 458.5 eV without the shoulder peak in the low energy region (FIG. 12C). The 2p _3/2 peak on the acid etched surface is slightly higher compared to the machined surface.

The acid-etched surface was chemically analyzed to identify the factors for its improved biological activity. The XPS spectrum reveals that the Cls peak decreases with the time of UV treatment, whereas the Ti2P and Ols peaks increase (FIGS. 12E, 12F, 12G). In particular, the shoulder peak at about 288 eV attributed to the oxygen-containing hydrocarbon was strongly absorbed and extinguished on the TiO ₂ surface. The atomic percentage of carbon continued to decrease from> 50% to <20% after 48 hours of UV irradiation (FIG. 12H). The least mean square approximation showed a negative linear correlation between the atomic ratio of carbon and the amount of albumin absorbed on the titanium surface with a high coefficient of determination (R ² = 0.930); The smaller the amount, the more albumin was absorbed on the surface (FIG. 12I). Osteoblast adhesion showed a different retrograde pattern; it increased exponentially with carbon removal (FIG. 12J). The contact angle was not particularly correlated with the rate of albumin absorption or cell adhesion (FIGS. 12K and 12L).

17. Effective use of a combination of UVA and UVC to bring the surface positively charged and attract cells As shown in FIG. 13, when combined with a UVA and UVC light source, compared to using UVA alone or UVC alone Cell adhesion was most increased.

References

Claims (32)

A medical implant having a metal surface, wherein the metal surface comprises a positively charged metal oxide. The medical implant according to claim 1, wherein the metal is selected from the group of titanium, platinum, tantalum, niobium, nickel, iron, chromium, cobalt, zirconium, aluminum and palladium. The medical implant of claim 1, wherein the metal surface is substantially free of hydrocarbons. The medical implant according to claim 1, wherein the implant comprises a carrier material. The medical implant according to claim 1, wherein the implant surface has a metal oxide cation. The medical implant according to claim 5, wherein the cation of the metal oxide is a titanium oxide cation. The medical implant according to claim 1, wherein the surface of the implant can attract proteins or cells at an increased rate. The medical implant according to claim 7, wherein the cells are selected from the group consisting of human mesenchymal stem cells and osteoblasts, and the protein is selected from the group consisting of bovine plasma albumin, fraction V, and bovine serum fibronectin. . The medical implant according to claim 7, wherein the protein and cells directly adhere to the implant surface. The medical implant according to claim 1, wherein the implant surface is capable of improving tissue-implant integration and / or bone-implant integration. The implant surface can increase protein absorption, increase osteoblast migration, increase osteoblast adhesion, increase osteoblast dispersion, increase osteoblast proliferation, increase osteoblast differentiation. The medical implant according to claim 1, which enables or a combination thereof. A method for imparting functionality to a medical implant, comprising: (1) providing a metal implant surface; and (2) treating the implant surface to charge the surface with a positive charge. How to have. 13. The method of claim 12, wherein the treated surface attracts proteins and / or cells at an enhanced rate. The method of claim 12, wherein the surface is a titanium surface. The method of claim 14, wherein the titanium surface comprises TiO ₂ . The method of claim 12, wherein the treated surface is substantially free of hydrocarbons. The method of claim 12, wherein the implant comprises a carrier material. 13. The method of claim 12, further comprising processing the implant prior to processing the implant, wherein the implant surface is processed by chemical etching, machining, or sand blasting. The method of claim 12, wherein the implant surface is treated with ultraviolet (UV) radiation. The method of claim 18, wherein the processed surface is treated with UV radiation. 21. The method of claim 19 or 20, wherein the UV radiation is at a wavelength selected from a group of about 170 nm to about 270 nm and about 360 nm to about 380 nm. 21. The method of claim 19 or 20, wherein the surface is treated with a combination of UV radiation at a wavelength of about 170 nm to about 270 nm and UV radiation at a wavelength of about 360 nm to about 380 nm. 21. A method according to claim 19 or 20, wherein the treatment with UV radiation takes up to 48 hours. The treatment with UV radiation is selected from the group of 30 seconds, 1 minute, 5 minutes, 15 minutes, 30 minutes, 1 hour, 3 hours, 5 hours, 10 hours, 15 hours, 24 hours, 36 hours and 48 hours. 24. The method of claim 22, wherein the method is performed in a time period. The method of claim 12, wherein the treated surface comprises a metal oxide cation. 26. The method of claim 25, wherein the metal oxide cation is a titanium oxide cation. 14. The method of claim 13, wherein the cells are selected from the group consisting of human mesenchymal stem cells and osteoblasts, and the protein is selected from the group consisting of bovine plasma albumin, fraction V, and bovine serum fibronectin. 14. The method of claim 13, wherein the protein and cells adhere directly to the implant surface. 13. The method of claim 12, wherein the treated implant surface can improve tissue-implant integration and / or bone-implant integration compared to an untreated implant surface. The method of claim 12, wherein the treated implant surface has improved bone forming ability compared to an untreated implant surface. The treated implant surface has increased protein absorption, increased osteoblast migration, increased osteoblast adhesion, increased osteoblast dispersion, increased osteoblast proliferation, osteoblast differentiation. 13. A method according to claim 12, which allows any increase or a combination thereof. 14. A method for improving bone-implant integration or osteogenesis capability comprising the method according to any of claims 11-13.

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